Vaccines have been very effective in protecting people from a wide variety of diseases, whether caused by viruses, bacteria, or fungi. The ability of vaccines to induce specific protection to such a wide range of pathogenic organisms results from their ability to stimulate specific humoral antibody responses, as well as cell-mediated responses. This invention relates to such vaccines, and particularly to a process for making conjugates, such as protein/polysaccharide conjugates, that are used in the preparation of vaccines and other valuable immunological reagents. The invention further relates to the vaccines and immunological reagents that are produced from the conjugates made in accordance with the invention.
Certain agents can stimulate an immune response with minimal chemical modifications, such as, for example, tetanus toxoid, which is immunogenic even in the absence of adjuvant. Other important agents are either non-immunogenic or poorly immunogenic, but they can be converted into immunogenic molecules or constructs, in which form they can induce vigorous immune responses. For example, most polysaccharides are poorly immunogenic. After they are coupled to proteins, however, the resulting construct becomes immunogenic. The conjugation of proteins to polysaccharides converts the polysaccharide from a weakly immunogenic T-cell independent antigen to a T-cell dependent antigen that recruits T-cell help, and thus stimulates heightened immune responses. Note the discussion by J. M. Cruse, et al. (Editors.), Conjugate Vaccines, Karger, Basel, (1989); and R. W. Ellis, et al. (Editors), Development and Clinical Uses of Haemophilus B Conjugate Vaccines, Marcel Dekker, New York (1994). These books are entirely incorporated herein by reference.
Conjugation of a protein and a polysaccharide may provide other advantageous results. For example, Applicant has found that a protein/polysaccharide conjugate enhances the antibody response not only to the polysaccharide component, but also to the protein component. This effect is described, for example, in the dual conjugate patent application of Mond and Lees, U.S. patent application Ser. No. 08/402,565 (filed Mar. 13, 1995, now U.S Pat. No. 5,585,100); application Ser. No. 08/444,727 (filed May 19, 1995, now abandoned); and application Ser. No. 08/468,060 (filed Jun. 6, 1995, now abandoned). These patent applications each are entirely incorporated herein by reference. This effect also is described in A. Lees, et al., "Enhanced Immunogenicity of Protein-Dextran Conjugates: I. Rapid Stimulation of Enhanced Antibody Responses to Poorly Immunogenic Molecules," Vaccine, Vol. 12, No. 13 (1994), pp. 1160-1166. This article is entirely incorporated herein by reference.
Techniques have been developed to facilitate coupling of proteins and polysaccharides. Note W. E. Dick, et al., "Glyconjugates of Bacterial Carbohydrate Antigens: A Survey and Consideration of Design and Preparation Factors," Conjugate Vaccines (Eds. Cruse, et al.,), Karger, Basel, 1989, beginning at page 48. This excerpt also is entirely incorporated herein by reference. Many techniques for activation of carbohydrates, however, are not suitable for use in aqueous media because the activating or functional reagents are not stable in water. For example, the use of N,N'-carbonyldiimidazole is described in Marburg et al., U.S. Pat. No. 4,695,624 (which patent is entirely incorporated herein by reference). This reagent must be used in organic media.
For use in aqueous media, applicant has developed the use of 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate, also called "CDAP" in this patent application, to activate polysaccharides. These activated polysaccharides may be directly or indirectly coupled to proteins. The use of CDAP is described in the following U.S. patent applications of Andrew Lees: U.S. patent application Ser. No. 08/124,491 (filed Sep. 22, 1993, now abandoned), U.S. patent application Ser. No. 08/408,717 (filed Mar. 22, 1995), and U.S. patent application Ser. No. 08/482,666 (filed Jun. 7, 1995). These U.S. patent applications each are entirely incorporated herein by reference. The use of CDAP also is described in Lees, et al., "Activation of Soluble Polysaccharides with 1-Cyano-4-Dimethylamino Pyridinium Tetrafluoroborate For Use in Protein-Polysaccharide Conjugate Vaccines and Immunological Reagents," Vaccine, Vol. 14, No. 3 (1996), pp. 190-198. This article also is entirely incorporated herein by reference.
Some polysaccharides have few or cryptic hydroxyls. Thus, these polysaccharides are not suitable for direct derivatization with vinylsulfone, nor for activation by other common methods, such as CNBr activation. Examples of such polysaccharides are Vi antigen and Neisseria meningiditis polysaccharide type C ("Neisseria PsC"). Additionally, some polysaccharides are pH sensitive. Thus, they are unsuitable for direct derivatization with vinylsulfone. Examples of such polysaccharides are Haemophilus influenzae type B ("PRP"), and Vi. Thus, the ability to perform the entire derivatization process at a lower pH may be important for derivatizing certain polysaccharides.
Often, however, the process of coupling a protein and a polysaccharide may lead to undesirable effects. In some cases, direct coupling can place the protein and polysaccharide in very close proximity to one another and encourage the formation of excessive crosslinks between the protein and the polysaccharide. Under the extreme of such conditions, the resultant material can become very thick (e.g., in a gelled state). Such a material would not be useful as a vaccine formulation.
Over-crosslinking also can result in decreased immunogenicity of the protein and polysaccharide components. In addition, the crosslinking process can result in the introduction of foreign epitopes into the conjugate or can otherwise be detrimental to production of a useful vaccine. The introduction of excessive crosslinks exacerbates this problem.
To limit the probability of excess crosslinking between the protein and polysaccharide, a spacer may be provided between the protein and polysaccharide. The spacer helps maintain physical separation between the protein and polysaccharide molecules, and it can be used to limit the number of crosslinks between the protein and polysaccharide. As an additional advantage, spacers also can be used to control the structure of the resultant conjugate. If a conjugate does not have the correct structure, problems can result that can adversely affect the immunogenicity of the conjugate material. The speed of coupling, either too fast or too slow, also can affect the overall yield, structure, and immunogenicity of the resulting conjugate product. Note Schneerson et al., Journal of Experimental Medicine, Vol. 152, beginning at pg. 361 (1980). This article is entirely incorporated herein by reference. Spacers help regulate the kinetics of the conjugation reaction.
In view of the potential advantages of using spacers, it is desirable to provide a process where a protein is coupled to a polysaccharide via a spacer. In this coupling procedure, spacers are used in the chemical reaction that is needed to join the protein with the polysaccharide. Spacers facilitate this chemical reaction by providing a functional group on one of the molecules that will react with a group present on the other molecule. Either the polysaccharide molecule or the protein molecule may be derivatized with the spacer molecule including the reactive functional group. If necessary, the other molecule also may be separately derivatized with a reactive functional group (e.g., a thiol, hydrazide, or amine) that will facilitate reaction with the spacer during conjugation.
The possible use of homobifunctional vinylsulfones has been considered for certain conjugation reaction processes. One member of this group is divinylsulfone, which has the following structure: ##STR1##
Divinylsulfone has been used to crosslink proteins and to derivatize proteins with haptens. Note, for example, "Conjugation to Preactivated Proteins Using Divinylsulfone and lodoacetic Acid," by Gunnar Houen, et al., Journal of immunological Methods, Vol. 181 (1995), pp. 187-200. This article is entirely incorporated herein by reference. The Houen article describes the coupling of a small protein (10 kDa) derivatized with divinylsulfone (DVS) to the lysines of a 45 kDa protein. Only low levels of protein coupling were observed. This article also describes the coupling of small haptens and peptides to highly derivatized DVS-protein. A large excess of the hapten was used in the described process. In this method, no effort was made to limit the degree of derivatization with divinylsulfone or to maintain the integrity of the protein. Indeed, in Houen, the goal was maximum derivatization of the protein.
Other researchers have described the use of divinylsulfone to couple proteins and haptens to solid phase gels with the purpose of obtaining affinity chromatography gels. See Porath, "General Methods and Coupling Procedures," Methods in Enzymology, Vol. 34 (1974), pgs. 13-30, and Porath et al,. "Immobilization of Enzymes to Agar, Agarose, and Sephadex Supports," Methods in Enzymology, Vol. 44 (1976), pgs. 19-45. These Porath documents also are entirely incorporated herein by reference. Note also, S. Pepper, "Some Alternative Coupling Chemistries for Affinity Chromatography," Molecular Biotechnology, Vol. 2 (1994), pp. 157-178. This article is entirely incorporated herein by reference. Problems with over-crosslinking and poor yield are described by Porath. Furthermore, these described methods for derivatizing with divinylsulfone required prolonged exposure to a high pH (pH 11). The combination of the multiplicity of the polysaccharide hydroxyl groups and the harsh reaction conditions promotes or induces over-crosslinking and aggregation of the polysaccharide. Such reaction conditions would be unsuitable for preparing soluble protein-polysaccharide conjugates.
The use of vinylsulfone derivatized polyethylene glycol ("PEG") to react with protein thiols and amines has been described by other researchers. See, for example, Morpurgo, et al., "Preparation and Characterization of Polyethylene Glycol Vinylsulfone," Bioconjugate Chemistry, Vol. 7 (1996), beginning at page 363 (which article is entirely incorporated herein by reference). The purpose of functionalizing with PEG, however, is to reduce the immunogenicity of the protein.
In addition to all of the above-noted problems in the reaction processes using divinylsulfone, other problems exist in using this material. In general, homobifunctional reagents, including divinylsulfone, have been found to produce a broad range of poorly defined conjugates. Note the discussion in G. T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, Calif., (1996), pg. 187. The entire Bioconjugate Techniques book is incorporated herein by reference.
In spite of these problems in using divinylsulfone, however, certain advantages exist for using this material. Divinylsulfone is a more universal linking reagent because it reacts with more nucleophiles as compared to iodoacetamides or maleimides. Other advantages of divinylsulfone relate to its availability, stability, water solubility, and cost. As compared to some agents used to derivatize proteins and/or polysaccharides, divinylsulfone is much less expensive and more readily available.